Challenges in Bioinformatics Workflows for Processing Microbiome Omics Data at Scale

The nascent field of microbiome science is transitioning from a descriptive approach of cataloging taxa and functions present in an environment to applying multi-omics methods to investigate microbiome dynamics and function. A large number of new tools and algorithms have been designed and used for very specific purposes on samples collected by individual investigators or groups. While these developments have been quite instructive, the ability to compare microbiome data generated by many groups of researchers is impeded by the lack of standardized application of bioinformatics methods. Additionally, there are few examples of broad bioinformatics workflows that can process metagenome, metatranscriptome, metaproteome and metabolomic data at scale, and no central hub that allows processing, or provides varied omics data that are findable, accessible, interoperable and reusable (FAIR). Here, we review some of the challenges that exist in analyzing omics data within the microbiome research sphere, and provide context on how the National Microbiome Data Collaborative has adopted a standardized and open access approach to address such challenges.

A unifying perspective of the ultrafast photo-dynamics of Orange Carotenoid Protein from Synechocystis: peril of high-power excitation, existence of different S* states and influence of tagging

A substantial number of Orange Carotenoid Protein (OCP) studies have aimed to describe the evolution of singlet excited states leading to the formation of photo-activated form, OCPR. The most recent one suggests that three picosecond-lived excited states are formed after the sub-100 fs decay of the initial S2 state. The S* state which has the longest reported lifetime of a few to tens of picoseconds is considered to be the precursor of the first red photoproduct P1. Here, we report the ultrafast photo-dynamics of the OCP from Synechocystis PCC 6803, carried out using Visible-NIR femtosecond time-resolved absorption spectroscopy as a function of the excitation pulse power and wavelength. We found that a carotenoid radical cation can form even at relatively low excitation power, obscuring the determination of photo-activation yields for P1. Moreover, the comparison of green (540 nm) and blue (470 nm) excitations revealed the existence of an hitherto uncharacterized excited state, denoted as S~, living a few tens of picoseconds and formed only upon 470 nm excitation. Since neither the P1 quantum yield nor the photo-activation speed over hundreds of seconds vary under green and blue continuous irradiation, this S~ species is unlikely to be involved in the photo-activation mechanism leading to OCPR. We also addressed the effect of His-tagging at the N- or C-termini on excited state photo-physical properties. Differences in spectral signatures and lifetimes of the different excited states were observed, at variance with the usual assumption that His-tagging hardly influences protein dynamics and function. Altogether our results advocate for careful consideration of the excitation power and His-tag position when comparing the photo-activation of different OCP variants, and beg to revisit the notion that S* is the precursor of photoactivated OCPR.

Visualizing polymeric components that define distinct root barriers across plant lineages

ABSTRACT Hydrophobic cell wall depositions in roots play a key role in plant development and interaction with the soil environment, as they generate barriers that regulate bidirectional nutrient flux. Techniques to label the respective polymers are emerging, but are efficient only in thin roots or sections. Moreover, simultaneous imaging of the barrier constituents lignin and suberin remains problematic owing to their similar chemical compositions. Here, we describe a staining method compatible with single- and multiphoton confocal microscopy that allows for concurrent visualization of primary cell walls and distinct secondary depositions in one workflow. This protocol permits efficient separation of suberin- and lignin-specific signals with high resolution, enabling precise dissection of barrier constituents. Our approach is compatible with imaging of fluorescent proteins, and can thus complement genetic markers or aid the dissection of barriers in biotic root interactions. We further demonstrate applicability in deep root tissues of plant models and crops across phylogenetic lineages. Our optimized toolset will significantly advance our understanding of root barrier dynamics and function, and of their role in plant interactions with the rhizospheric environment.

First person – Ajay Labade and Adwait Salvi

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Ajay Labade and Adwait Salvi are co-first authors on ‘ Nup93 and CTCF modulate spatiotemporal dynamics and function of the HOXA gene locus during differentiation’, published in JCS. Ajay is a postdoctoral researcher in the lab of Dr Jason Buenrostro at Harvard University, Cambridge, USA, where he develops multimodal genomics technologies for the investigation of functional genome organization. Adwait is a PhD student in the lab of Filippo Rijli at Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland, who works on deciphering the role of 3D genome organization in gene regulation during differentiation.

NuMA regulates mitotic spindle assembly, structural dynamics and function via phase separation

A functional mitotic spindle is essential for accurate chromosome congression and segregation during cell proliferation; however, the underlying mechanisms of its assembly remain unclear. Here we show that NuMA regulates this assembly process via phase separation regulated by Aurora A. NuMA undergoes liquid-liquid phase separation during mitotic entry and KifC1 facilitates NuMA condensates concentrating on spindle poles. Phase separation of NuMA is mediated by its C-terminus, whereas its dynein-dynactin binding motif also facilitates this process. Phase-separated NuMA droplets concentrate tubulins, bind microtubules, and enrich crucial regulators, including Kif2A, at the spindle poles, which then depolymerizes spindle microtubules and promotes poleward spindle microtubule flux for spindle assembly and structural dynamics. In this work, we show that NuMA orchestrates mitotic spindle assembly, structural dynamics and function via liquid-liquid phase separation regulated by Aurora A phosphorylation.

Inferring kinetic rate constants from single-molecule FRET trajectories – a blind benchmark of kinetic analysis tools

Single-molecule FRET (smFRET) is a versatile technique to study the dynamics and function of biomolecules since it makes nanoscale movements detectable as fluorescence signals. The powerful ability to infer quantitative kinetic information from smFRET data is, however, complicated by experimental limitations. Diverse analysis tools have been developed to overcome these hurdles but a systematic comparison is lacking. Here, we report the results of a blind benchmark study assessing eleven analysis tools used to infer kinetic rate constants from smFRET trajectories. We tested them against simulated and experimental data containing the most prominent difficulties encountered in analyzing smFRET experiments: different noise levels, varied model complexity, non-equilibrium dynamics, and kinetic heterogeneity. Our results highlight the current strengths and limitations in inferring kinetic information from smFRET trajectories. In addition, we formulate concrete recommendations and identify key targets for future developments, aimed to advance our understanding of biomolecular dynamics through quantitative experiment-derived models.

Mitochondria-Shaping Proteins and Chemotherapy

The emergence, in recent decades, of an entirely new area of “Mitochondrial dynamics”, which consists principally of fission and fusion, reflects the recognition that mitochondria play a significant role in human tumorigenesis and response to therapeutics. Proteins that determine mitochondrial dynamics are referred to as “shaping proteins”. Marked heterogeneity has been observed in the response of tumor cells to chemotherapy, which is associated with imbalances in mitochondrial dynamics and function leading to adaptive and acquired resistance to chemotherapeutic agents. Therefore, targeting mitochondria-shaping proteins may prove to be a promising approach to treat chemotherapy resistant cancers. In this review, we summarize the alterations of mitochondrial dynamics in chemotherapeutic processing and the antitumor mechanisms by which chemotherapy drugs synergize with mitochondria-shaping proteins. These might shed light on new biomarkers for better prediction of cancer chemosensitivity and contribute to the exploitation of potent therapeutic strategies for the clinical treatment of cancers.

Nup93 and CTCF modulate spatiotemporal dynamics and function of the HOXA gene locus during differentiation

Nucleoporins regulate nuclear transport and are also involved in DNA damage, repair, cell cycle, chromatin organization, and gene expression. Here, we studied the role of nucleoporin Nup93 and the chromatin organizer CTCF in regulating HOXA expression during differentiation. ChIP sequencing revealed a significant overlap between Nup93 and CTCF peaks. Interestingly, Nup93 and CTCF are associated with the 3' and 5′HOXA genes respectively. Depletions of Nup93 and CTCF antagonistically modulate expression levels of 3′and 5′HOXA genes in undifferentiated NT2/D1 cells. Nup93 also regulates the localization of the HOXA gene locus, which disengages from the nuclear periphery upon Nup93 but not CTCF depletion, consistent with its upregulation. The dynamic association of Nup93 and CTCF with the HOXA locus during differentiation correlates with its spatial positioning and expression. While Nup93 tethers the HOXA locus to the nuclear periphery, CTCF potentially regulates looping of the HOXA gene cluster in a temporal manner. In summary, Nup93 and CTCF complement one another in modulating the spatiotemporal dynamics and function of the HOXA gene locus during differentiation.